RECENT FINDINGS: [unreadable] THE FUNCTION AND STRUCTURE OF ORF1p. ORF1p is a trimer, the monomer of which has three domains: A rapidly evolving amino terminal domain of unknown function; a coiled coil domain, which is required for trimer formation and which also evolves rapidly having undergone several episodes of adaptive evolution; and a highly conserved carboxyl domain that binds nucleic acids. These structural and evolutionary characteristics of ORF1p are typical of all L1 mammalian ORF1ps examined to date. Thus, rapid evolution of the amino terminal half of ORF1p may be essential for the persistence of L1 activity during the evolution of mammals. Therefore, we are correlating the effects of evolutionary changes in ORF1p on its retrotransposition activity and a number of other properties: e.g., RNA-binding, trimer formation, nucleic acid chaperone activity (each of which is essential for retrotransposition), and interaction with host proteins (see Z01 DK057811-01 LMCB: Mammalian L1 Retrotransposon - host interaction). We constructed an ancestral version of ORF1p (L1Pa5) that predated the evolutionary changes of the modern version (L1Pa1) and mosaic ORF1ps that contain modern and ancestral regions. Among our recent findings are: The amino terminal domain is absolutely essential for retrotransposition but not for trimer formation or interaction with host proteins. Substitution of just a few ancestral amino acids for their modern counterparts in the coiled coil domain completely abolishes retrotransposition but has no measurable effect on trimer formation. As the structure of these proteins will be of considerable interest, especially because ORF1p is not a member of any known protein family, we are collaborating with Fred Dyda and Alison Hickman, structural biologists in NIDDK to determine their structures. Initial experiments showed that expression of ORF1p in E. coli was beset by aberrant translational initiation leading to contamination of trimers by truncated monomers. However, these experiments did show that the purified protein was sufficiently soluble for crystallization. We are now using baculovirus infection to express the protein in insect cells to bypass the aberrant initiation during protein synthesis. [unreadable] DEVELOPMENT OF A NEW RETROTRANSPOSITION ASSAY. Retrotransposition assays generally rely on the detection of a DNA copy (reporter gene) of a spliced RNA transcript. The requirement for RNA splicing ensures that the DNA copy has gone through an RNA intermediate, a hallmark of retrotransposition. There are two major problems with the current retrotransposition assay: First, the splicing reaction occurs via the normal splicesomal pathway, a pathway the L1 would not normally encounter as L1 RNA is not spliced. Cytoplasmic RNAs that have passed through the splicesomal pathway bind a number of proteins unique to this pathway. Thus, an L1 RNP replication complex assembled from an L1 RNA transcript that has traversed the splicesomal pathway could contain host factors that it normally does not. We rectified this problem by substituting a self-splicing intron for the splicesomal intron. Although the two vectors generate identical inserts, they differ in their retrotransposition activity. The second problem with the current assay is that the integrity of the L1 3 UTR RNA is severely compromised by the presence of the reporter gene for retrotransposition. The reporter gene encodes (G418) resistance and contains along with an intron a promoter and transcriptional stop signals. Together these sequences account for about 2 kb of DNA all of which is co-transcribed within or close to the L1 3UTR. Thus, any role of the highly conserved sequences and sequence motifs present in the 200 bp 3 UTR RNA could be overwhelmed by its juxtaposition to 2000 bp of extraneous RNA. We are now rectifying this problem by replacing the reporter gene with a 15 bp sequence split by the self-splicing (autocatalytic) intron. Thus, as soon as the transcript is synthesized, the intron is spliced out generating an L1 transcript that differs from a normal L1 transcript by only 15 nucleotides. By inserting the reporter in the least conserved region of the 3 UTR we hope to minimize its effect on L1 RNA. We will isolate any retrotransposition products generated from our reporter transcripts by hybridization with PNA (a nucleic acid polymer in which the phoshpodiester backbone is replace by amide bonds) and then quantify the products by real time (RT) PCR. We are collaborating in this effort with Dr. Daniel Appella (NIDDK) who has developed modifications to PNA synthesis that greatly increase both its sensitivity and specificity.